Apolipoprotein (apo) E4 has been identified as a major risk factor or susceptibility gene for the development of Alzheimer's disease and other neurodegenerative disorders. Several hypotheses have been advanced to explain this association, including its role in altering amyloid beta (Abeta) peptide clearance and/or deposition (plaque formation), altering tau phosphorylation and microtubule biology (tangle formation), and altering cytoskeletal structure and function (remodeling and repair). Thus, apoE3 and apoE4 may impact neurobiology and neuropathology through multiple processes or pathways. In fact, it is reasonable to speculate that under various physiological or pathological conditions apoE may play different roles, some of which undoubtedly remain to be elucidated. Recent preliminary studies have extended our understanding of isoform-specific effects of apoE on neurons and may shed light on additional unique roles for apoE in neurobiology. First, apoE4 stimulates Abeta production in neuronal cells to a greater extent than apoE3 and the enhanced Abeta production is associated with increased amyloid precursor protein recycling. Sccond, apoE4 potentiates Abeta-induced lysosomal leakage and cell death whereas apoE3 does not. Third, apoE4 undergoes proteolytic cleavage to form intracellular carboxyl-terminal-truncated forms of apoE that induce neurofibrillary tangle-like inclusion formation in neurons (apoE4 > apoE3). Transgenic apoE4 mice expressing apoE in neurons have increased levels of tangle-like structures and phosphorylated tau inclusion bodies in neurons. The goal of this project is to investigate the mechanisms whereby apoE3 versus apoE4 alters these cellular pathways, including the apoE isoform-specific effects on Abeta production (Aim 1), Abeta-induced lysosomal leakage (Aim 2), and the formation of neurofibrillary tangle-like inclusions in neurons associated with the generation of bioactive intracellular proteolytic fragments of apoE (Aim 3). We will determine how these cellular pathways are modulated by the unique structural (conformational) characteristics of apoE, including domain interaction, in which the amino- and carboxyl-terminal domains interact only in apoE4 (arginine-61 reacting with glutamic acid-255) and molecular instability, in which apoE4 more readily forms reactive folding intermediates than apoE3. These studies, involving both in vitro and in vivo approaches, will provide additional insights into the role of apoE4 and may identify new therapeutic targets for apoE4-associated neuropathological disorders.